Bone morphogenic protein-2 (BMP-2)-conjugated three-dimensional (3-D)-printed poly (L-lactic acid)(PLLA) scaffold is likely promising as an effective bone substitute for enhancing bone regeneration of massive bone defects caused by tumor resection, traumatic injury, or congenital diseases. The authors developed a new bone substitute using a novel strategy composed of 3-D-printed PLLA scaffolds through a sequential coating of catechol-conjugated alginate (C-AL), BMP-2, and collagen (CO). The 3-D-printed PLLA scaffold was successfully obtained with 5 mm of diameter, 1 mm of thickness, 400 lm of pore size, 187–230 lm of grid thickness, and 82% of porosity. Alkaline phosphatase (ALP) activity of the BMP-2-immobilized PLLA scaffold in MC3T3-E1 and W-20-17 cells was more increased than BMP-2 itself due to the controlled release of BMP-2 from the scaffold. Tenfold new bone formation for the BMP-2-immobilized PLLA scaffold was obtained by micro-CT analysis than PLLA scaffold without BMP-2 weeks after 4 weeks of transplantation model mouse. Further another big animal model study should be performed before clinical trials.

Human palatine tonsil-derived mesenchymal stem cells (TMSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of TMSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of TMSCs and to access the differential effects of transforming growth factor beta 3 (TGF-β3) on the tenogenesis of TMSCs. Human tonsil was obtained after tonsillectomy. Using a cytometric analysis, we were able to find that the TMSCs had typical mesenchymal stem cell markers: positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45. Using TGF-β3, the expressions of tenocyte-specific genes and proteins, such as collagen type 1 (COL1), tenomodulin (TNMD), and scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR), immunofluorescence staining, immunohistochemistry and Western blot analyses. Quantitative PCR assay showed that TGF-β3 significantly increased the expressions of tenocyte lineage marker genes, including COL1, TNMD, and SCX, at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/mL at a 7-day exposure to TGF-β3. Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-β3-treated TMSCs in a 3-day treatment, which were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. This study demonstrated that TMSCs in tenogenic stimulation with TGF-β3 have a high tenogenic differentiation potential.
Blotting, Western ; Collagen ; Fluorescent Antibody Technique ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells* ; Palatine Tonsil ; Polymerase Chain Reaction ; Stem Cells ; Tonsillectomy ; Transforming Growth Factor beta

A 52-year-old man was referred to our hospital due to fever and myalgia that occurred 2 weeks earlier. He showed a complete atrioventricular block on his electrocardiogram, and his vital signs were unstable. On his transthoracic echocardiograph, the 1.5 cm vegetation in the aortic valve with severe aortic regurgitation suggested infective endocarditis. His transesophageal enchocardiograph showed abscess in his mitral-aortic intervalvular fibrosa and vegetation was suspected on his anterior mitral valve leaflet. The patient underwent an emergent operation for valve replacement with temporary epicardial pacing. Intraoperatively, the septal leaflet of his tricuspid valve was injured during the debridement of the abscess pocket that was extended to the membranous septum. The aortic, mitral, and tricuspid mechanical valves were replaced with annular reconstruction without complications. After 14 days of intravenous antibiotics, we successfully changed the epicardial pacemaker into a transvenous DDD-type permanent pacemaker by placing a left ventricular lead via the coronary sinus and an atrial lead in the right atrium appendage. The patient was discharged in a tolerable state and was examined uneventfully in our hospital's outpatient clinic for 8 months.
Abscess ; Ambulatory Care Facilities ; Anti-Bacterial Agents ; Aortic Valve ; Aortic Valve Insufficiency ; Atrioventricular Block* ; Coronary Sinus* ; Debridement ; Electrocardiography ; Endocarditis* ; Fever ; Heart Atria ; Heart Block ; Heart Valve Prosthesis ; Humans ; Middle Aged ; Mitral Valve ; Myalgia ; Pacemaker, Artificial ; Tricuspid Valve ; Vital Signs

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5’-serially deleted eNOS promoters revealed that Tax-responsive element site, located at −962 to −873 of the eNOS promoter, was respon-sible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominantnegative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser133 level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser1981 and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473 , p-CREB-Ser133 , eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser1981 , p-Akt-Ser473 , p-CREB-Ser 133 , and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

Platelet membrane receptor glycoproteins (GP) are essential for the platelet activation process, and the genetic polymorphisms in the genes that encode platelet glycoproteins have been proposed to influence the risk of acute coronary syndrome and atherosclerosis. In this study, we investigated the role of GPIa, HPA-1 and HPA-3 polymorphisms as putative risk factors for myocardial infarction (MI) and the extent of coronary artery disease. We selected 1, 073 subjects who underwent coronary angiography; 242 had normal or minimal coronary atherosclerosis, and 831 patients had significant coronary artery disease (CAD). The genotype was determined by the methods of single base extension for C807T/G873A polymorphisms of GPIa, and restriction fragment length polymorphism for HPA-1 and HPA-3. The C807T and G873A polymorphisms of GPIa showed complete linkage in the Korean population. For HPA-1 gene polymorphism, only the HPA-1a/a (PlA1/A1) genotype was observed in 192 selected subjects from our study population. The distribution of GPIa (C807T/G873A) and HPA-3 genotypes did not differ significantly between normal subjects and CAD subjects. No significant association between MI and both gene polymorphisms was present. However, for the subgroup analysis of young male patients whose age was less than 56 years, the genotype frequency of HPA-3b/b was significantly lower in patients with MI compared to patients without a history of MI (7.5% vs. 20.0%, p=0.04). The odds ratio for HPA-3 b homozygosity versus the HPA-3a carrier was 0.32 (95% CI, 0.10- 0.99, p=0.04). Conclusively, HPA-3 polymorphism was associated with MI in Korean individuals younger than 56 years of age, but other polymorphisms of GP, which we studied, were not associated with both the extent of coronary atherosclerosis or MI.
Aged ; Coronary Arteriosclerosis/epidemiology/*genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease/epidemiology ; Genotype ; Human ; Integrin alpha2/*genetics ; Integrin beta3/*genetics ; Korea ; Male ; Middle Aged ; Myocardial Infarction/epidemiology/*genetics ; Platelet Membrane Glycoprotein IIb/*genetics ; *Polymorphism (Genetics) ; Risk Factors ; Support, Non-U.S. Gov't

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The delta delta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.
Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis/*methods ; Female ; Genetic Predisposition to Disease/epidemiology ; Genetic Screening/*methods ; Heterozygote ; Heterozygote Detection/methods ; Humans ; Korea/epidemiology ; Male ; Middle Aged ; Muscular Atrophy, Spinal/*epidemiology/genetics/*metabolism ; Nerve Tissue Proteins/*genetics ; Polymorphism, Genetic ; *Quantitative Trait, Heritable ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Risk Assessment/*methods ; Risk Factors

BACKGROUND: Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments.METHODS: TMSCs were preconditioned with Dulbecco's Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4 days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers.RESULTS: Preconditioning TMSCs in EGM for 4 days and then replating them on Matrigel induced the formation of a capillary-like network in 3 h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells.CONCLUSION: This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4 days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.
Blood Vessels ; Blotting, Western ; Eagles ; Endothelial Cells ; Fluorescent Antibody Technique ; Gelatin ; Genome ; Human Umbilical Vein Endothelial Cells ; Mesenchymal Stromal Cells ; Morphogenesis ; Nitric Oxide Synthase Type III ; Palatine Tonsil ; Real-Time Polymerase Chain Reaction ; Serine ; Stem Cells